1. Field of the Invention
The present invention relates to an expression vector containing lectin gene regulation site of mud loach for producing tralsgenic fishes. More particularly, it relates to an expression vector of mud loach or carp growth hormone gene fused to lectin gene regulation site of mud loach, and a method of producing a fast-growing transgenic mud loach or carp by transforming it with the expression vector, and a transgenic mud loach or carp produced thereby.
2. Description of the Related Art
Transgenesis of fish is considered to provide a useful model for the research in expression and regulation of vertebrate genes and to produce a novel fish strain having a useful function; thereby to overcome the limit of productivity increase which remains unsolved in the conventional fish breeding (Iyengar, A., Muller, F. and Maclean, N., 1996. Regulation and expression of transgenes in fish a review. Transgenic Research 5, 147-166; Dunham, R. A., Devlin, R. H., 1999. Comparison of traditional breeding and transgenesis in farmed fish with implications for growth enhancement and fitness. In: Murray, J. D., Anderson, G. B., Oberbauer, A. M., McGloughlin, M. N. (Eds), Transgenic animals in agriculture. CAB International, Wallingford, UK, pp. 209-229). In an early stage of fish transgenesis, mammal or microorganism gene regulation sites are used to produce transgenic fishes. However, since the mammal or microorganism gene regulation sites were known to have little effect on fish cells, many efforts have been made to acquire new fish-originated promoters for developing transgenic fish.
Most of efforts to produce transgenic fishes for increasing productivity have been concentrated on the improvement of growth rate due to the recombination of growth hormone gene. However, for all the attempts at various fishes, successful improvements of growth rate have been limited to several fishes. Further, those fish-originated promoters to be used in fish transgenesis have been also limited to several species and applications. Up to this time, successful examples of improvement of growth rate and transformation have been transfer of the MT promoter of sockeye salmon and AFP promoter of ocean pout fused with salmon growth hormone gene into salmon species (Devlin, R. H., Yesaki, T. Y., Biagi, C. A., Donaldson, E. M., Shan, R. M., 1994. Extraordinary salmon growth. Nature 371, 209-210; Devlin, R. H., Yesaki, T. Y., Donaldson, E. M., Du, S. J., Hew, C. L., 1995. Production of germline transgenic Pacific salmonids with dramatically increased growth performance. Canadian Journal of Fisheries and Aquatic Science 52, 1376-1384; Hew, C. L., Fletcher, G. L. and Davies, P. L., 1995. Transgenic salmon: tailoring the genome for food production. Journal of Fish Biology 47, 1-19) or into tilapia (Rahman, M. A., Mak, R., Ayad, H., Smith, A., and Maclean, N., 1998. Expression of a novel piscine growth hormone gene results in growth enhancement in transgenic tilapia (Orechromis niloticus). Transgenic Research 7, 357-369), transfer of CMV promoter fused with growth hormone gene cDNA of tilapia into tilapia (Martinez, R., Estrada, M. P., Berlanga, J., Guillen, I., Hernandez, O., Cabrera, E., Pimentel, R., Morales, R., Herrera, F., Morales, A., Pina, J. C., Abad, Z., Sanchez, V., Phillippa, M., Lleonar, R. and Fuente, J., 1996. Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone. Molecular Marine Biology and Biotechnology 5, 62-70), transfer of xcex2-actin promoter of carp fused with growth hormone gene cDNA of carp into carp (Hinits, Y. and Moav, B., 1999. Growth performance studies in transgenic Cyprinus carpio. Aquacutlure 173, 285-296), and transfer of expression vector of mud loach growth hormone containing xcex2-actin promoter of mud loach into mud loach (Nam, Y. K., Noh, J. K., Cho, Y. S., Cho, H. J., Cho, K. N., Kim, C. G. and Kim, D. S., 2001. Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach (Misgurnus mizolepis). Transgenic Research 10, 353-362). Except the success of virus-originated CMV promoter transferred into tilapia, promoters and regulation sites of the same or related fish species have been used as expression vectors of growth hormone.
In these successful examples, however, the effects cover a wide range from a limited improvement of growth rate to a greatly accelerated growth rate by dozens of times according to the expression vector and fish species to be used. Further, some promoters have been too effective to result in physiological deformation and adverse effect caused by an excessive expression of growth hormone (Dunham, R.A., Devlin, R.H., 1999).
A mud loach (Misgurnus mizolepis), a representative fresh-water fish species in Korea, has been widely used as an excellent food in Korea and Japan and the scale of its market and consumption is extending. However, due to a contamination of natural basin system and reclamation of rivers, the amount of natural catch decreases every year. Accordingly, culturing of mud loaches becomes inevitable and improvement of its breed through transgenesis is required to increase the productivity in a short time.
A possibility of transforming mud loach has been recognized when an expression vector of mud loach growth hormone gene containing xcex2-actin gene regulation site of mud loach was transferred into a fertilized egg to show a great growth acceleration effect However, since the xcex2-actin promoter to be used for the transgenesis has an excessive activity to various tissues, some of transgenic mud loaches show an excessive growth acceleration and grow to an unnecessary large size; therefore, more stable expression vector is required to be developed (Nam et al., 2001).
A carp (Cyprinus carpio) is one of fishes to be cultured widely in the world containing Korea. For all the attempts to improve carp breeds all over the world, a great increase of productivity is considered to be difficult for some time. Accordingly, transgenic carp of high growth rate are inevitable to increase productivity.
Research in transgenesis targeted on a carp has been made by some of groups. For example, Fu et al. led to a fast-growing carp by using an expression vector containing human growth hormone gene fused with mouse MT promoter (Fu, C., Cui, Y, Hung, S. S. O. and Zhu, Z., 1998. Growth and feed utilization by F4 human growth hormone transgenic carp fed diets with different protein levels. Journal of Fish Biology 53, 115-129), which had only slight improvement of growth rate and gave unwillingness about the genes and regulation sites originated from human. Further, Hinits and Moav (Hinits, Y. and Moav, B., 1999) tried to transform a carp by using carp xcex2-actin promoter and carp growth hormone cDNA, which showed also lower improvement of growth rate than two times except the result of winter period experiment. Therefore, a transgenic carp expected to improve the productivity greatly has not yet been developed.
Considering the above, a transgenesis of mud loach or carp is required to improve their culturing productivity, and therefore, a demand for developing an expression vector which expresses the transferred genes in an effective and stabilized marmer in mud loach or carp and gene regulation site therefor is increasing.
To improve the culturing productivity by producing a fast-growing transgenic mud loach or carp, it is an object of the present invention to provide a gene regulation site which can express the genes transferred into a mud loach or carp in an effective and stabilized manner, and an expression vector containing the gene regulation site.
It is another object of the present invention to provide a fast-growing mud loach and carp transformed with the expression vector, and a method of producing a fast-growing transgenic mud loach and carp using the expression vector.
To achieve the object of the present invention, there is provided an isolated polynucleotide comprising a lectin gene regulation site of a mud loach, expressed as SEQ ID NO: 1.
In accordance with a further aspect of the present invention, there are provided an expression vector comprising a lectin gene regulation site of a mud loach, an expression vector comprising a lectin gene regulation site of a mud loach and a growth hormone gene of a mud loach, and an expression vector comprising a lectin gene regulation site of a mud loach and a growth hormone gene of a carp.
In accordance with still another aspect of the present invention, there are provided a method of making a transgenic mud loach or carp comprising microinjecting the expression vector into fertilized eggs of a mud loach or carp and culturing the eggs such that the eggs hatch and result in a mud loach or carp fish which expresses the growth hormone gene at levels which increase the rate of growth of the fish relative to wild-type mud loach or carp, and a mud loach or carp transformed with the expression vector.
In the present invention, a lectin gene and regulation site containing its promoter is isolated from a mud loach, and then the lectin promoter is fised with various structural genes (e.g., genes for coding such reporter proteins as CAT or BFP) to construct reporter expression vectors, which confirm the ability of the lectin promoter to induce expressions in vivo. Especially, an expression vector of growth hormone gene containing the lectin gene regulation site of a mud loach is transferred into a mud loach or carp to produce a fast-growing transgenic fish breed, thereby increasing culturing productivity.